KR20160058934A - Methods and apparatus for delivery of molecules across layers of tissue - Google Patents

Abstract

Exemplary methods of opening a pore to move a molecule into a tissue, including applying a plasma to a tissue surface and applying a carrier comprising the one or more molecules to the surface of the tissue, are disclosed herein.

Description

≪ Desc / Clms Page number 1 > METHODS AND APPARATUS FOR DELIVERY OF MOLECULES ACROSS LAYERS OF TISSUE < RTI ID =

<Cross reference of related literature>

This regular utility patent application claims priority and benefit from U.S. Provisional Patent Application 61 / 883,701, entitled " Method and Apparatus for Delivering Molecules Across the Skin Layer, " filed September 27, 2013. This application is incorporated herein by reference in its entirety.

<Technical Field>

The present invention relates generally to methods and solutions for enabling or enhancing intracellular or intracellular molecule transport across tissue comprising a skin layer using non-thermal plasma, To open up tissue to the tissues and deep tissue hygiene treatment; Delivery of vaccines, drugs, and cosmetics; Improvement of skin health; For transporting one or more molecules across the skin or tissue layer.

Transdermal delivery is localized, noninvasive, has the potential for sustained and controlled drug delivery and other molecular excretion. In addition, transdermal drug delivery avoids first-pass metabolism, which reduces drug concentration before the drug reaches the circulatory system. In addition, transdermal absorption minimizes the risk of gastrointestinal irritation and minimizes other complications associated with pain and parenteral administration.

Transdermal delivery, however, requires the molecule to pass through the skin. Figure 1 shows layers of skin 100. The outer layer of skin 100 is the stratum corneum ("SC") 102. SC (102) is composed of dead, flat, keratin-rich cells called keratinocytes. These dense cells are surrounded by a complex mixture of intercellular lipids - ceramides, free fatty acids, cholesterol and cholesterol sulfate. The prominent diffuse pathway of the molecule across the SC appears to be intercellular. The remaining skin layers are epidermis (viable epidermis) 104, dermis 106, and subcutaneous tissue 108.

Since the skin 100 has a barrier property to prevent skin permeation of the molecules, i.e. SC 102 which is very lipophilic, only a small percentage of the compound can be delivered percutaneously. As a result, only molecules having a molecular weight (MW) of less than 500 daltons can be administered locally or percutaneously. Often, in pharmaceutical applications, the development of innovative compounds is limited to less than 500 Daltons of MW when topical dermatological treatment, percutaneous systemic therapy or vaccination is the goal. In addition, most drug delivery across the skin is very slow, and the delay time to reach steady-state flux is measured in hours. Achieving a therapeutically effective drug level is therefore difficult without artificially promoting skin penetration.

Several chemical and physical enhancement techniques have been developed as attempts to reversibly compromise skin barrier function. These attempts can be classified into passive and active methods.

Passive methods for enhancing transdermal drug delivery include use of vehicles such as ointments, creams, gels, and passive patching techniques. In addition, other passive methods of artificially damaging the barrier to allow improved penetration of the active material, such as, for example, the use of a small There are microneedles to create the work. Since the skin barrier properties are fundamentally unchanged, the amount of material that can be delivered using these methods is limited.

Active methods for promoting transdermal drug delivery systems involve the use of external energy to act as a driving force and / or to act to reduce the SC barrier resistance and promote penetration of drug molecules into the skin. Iontophoresis and electroporation are two common methods of active transdermal drug delivery systems.

Iontophoresis is a process that increases the penetration of electrically charged drugs into the skin by the application of electrical current. The amount of compound delivered is directly proportional to the amount of charge past; That is, it depends on the current applied, the current application duration and the skin surface area in contact with the active electrode. The advantage of the iontophoresis method is that it includes an improved start time and also a faster offset time - that is, when the current is turned off, there is no further transport of the compound.

For drug delivery using iontophoresis, the drug is applied under the electrode with the same charge as the drug and the return electrode with the opposite charge and placed on the body surface. The current below the patient's threshold limit level is applied for the appropriate length of time. Because the same charges repel each other, the electrical current increases the penetration of the drug into the surface tissue without changing the SC structure. The iontophoresis method transports the drug primarily through pathways present in the skin, such as through the hair follicles and glands. The iontophoresis method is typically used when a low level of delivery is desired over a long period of time. The iontophoresis involves the use of relatively low transdermal voltages (<100 V).

Percutaneous absorption of drugs through iontophoresis is affected by drug concentration, drug polarity, pH of the donor solution, ionic competition, ionic strength, electrode polarity, and so on. Iontophoresis has safety problems that can cause patient discomfort, muscle contractions, pain and sometimes even skin damage and burns, due to the use of electrical contact with the skin.

Electroporation is a method for transdermal drug delivery consisting of applying a high voltage pulse to the skin. The applied high voltage plays a dual role. First, it creates a new pathway for enhancing drug permeability, and second, it provides an electrical force that moves equally charged molecules to newly created pores. Electroporation is usually used for unilamellar phospholipid bilayers in cell membranes. However, electroporation of skin has proven possible even if SC contains multilamellar, intercellular lipid bilayers with phospholipids and does not contain living cells.

Electroporation of the skin requires a high transdermal voltage (~ 100 V or more, usually> 100 V). In percutaneous electroporation, a prominent voltage drop of the electric pulse applied to the skin occurs across the SC. This voltage distribution causes an electrical breakdown of the SC (electroporation). If the voltage of the applied pulse exceeds the voltage limit of about 75-100 V, a microchannel or "local transport zone" is created through the breakdown region of the SC.

DNA introduction is the most common use for electroporation. Electroporation of isolated cells also includes (1) introduction of biochemical reagents for enzyme, antibody, and other intracellular assays; (2) selective biochemical loading of one size cell in the presence of many small cells; (3) introduction of viruses and other particles; (4) cell death under non-toxic conditions; And (5) insertion of macromolecules into the cell membrane.

The presence of an electrode in contact with the skin / tissue and the delivery of current into the skin / tissue in this manner leads to patient discomfort, muscle contraction, pain, and sometimes even skin damage and burns. In addition, electroporation often takes time, e.g., 6 to 24 hours, to transfer a therapeutic dose of the drug or other molecules percutaneously.

U.S. Patent No. 8,455,228 entitled " Method For Facilitating Electroporation Using Direct Transfer and Charged Steam "describes a method and apparatus according to the present invention that uses an electric field to control the electrochemical potential of a target molecule And thus provides molecular transport of the target molecule into and / or across the tissue by diffusive transport mechanisms. " The '228 patent discloses the first embodiment with dielectric properties to ensure that it will have enough charge to polarize charged objects contained within the vessel and a plurality of electroporation applicators. The contents of the '228 patent are subject to various drawbacks. First, it requires that the molecule be polarizable or chargeable; second, it requires an electroporation applicator; and third, the molecules are in contact with the plasma during the process, which can change the molecular structure causing adverse outcomes.

The '228 patent also discloses a second embodiment that utilizes a plasma jet with a ground ring around the inner chamber. The disclosure includes cells suspended in a fluid in an inner chamber and inflow into cells; Or &lt; / RTI &gt; intradermally injecting the plasmid and facilitating exposure of the injection site to the plasma.

U.S. Patent Publication No. 2014/0188071 discloses a method of applying a substance to the skin and applying the plasma to the same region. The '071 publication discloses an open cell foam that holds a drug, water, and the like and applies the plasma through an open cell foam. Applying the plasma through the open cell foam and contacting the drug with the plasma can alter the molecular structure of the drug and make the unwanted side effects and / or drugs ineffective.

U.S. Patent Publication No. 2012/0288934 discloses a plasma jet in which the active material is applied to the skin along with the gas stream of the plasma jet and transported to the living cell area through the barrier door opened by the plasma. Applying the active material along with the gas stream of the plasma jet can alter the molecular structure of the active material and make the undesired side effects and / or active material ineffective.

Applying a plasma to the skin surface to open the pores in the skin; Applying to the skin surface a carrier having at least one molecule having a molecular weight of greater than 500 Da; And methods for delivering or transferring molecules into the skin, including transporting the molecules through pores to a desired depth.

Additionally applying a plasma to the surface of the skin to open the pores in the skin; Applying to the skin surface a carrier having at least one molecule having a molecular weight of less than 500 Da; And methods of enhancing penetration by transporting molecules through the pores to a desired depth are disclosed herein.

Additionally applying a plasma to the skin surface to open the pores of the skin; Applying a carrier having at least one molecule having a molecular weight of greater than 500 Da to the skin surface for a predetermined period of time; And then applying the plasma again.

Exemplary methods of applying bactericides to the skin are disclosed herein. Exemplary methods include applying a plasma to the surface of the skin to open the pores in the skin; Then applying a sterilizing agent to the surface of the skin; And transporting the disinfectant to a desired depth through the pores.

Exemplary methods of transdermal drug delivery are disclosed herein. Exemplary methods include applying a plasma to the skin surface to open the pores of the skin; Then applying the drug to the surface of the skin; And transporting the drug to the desired depth through the pore.

An exemplary method of transdermal vaccination is disclosed herein. Exemplary methods include applying a plasma to the skin surface to open the pores of the skin; Then applying the vaccine to the skin surface; And transporting the vaccine to the desired depth through the pore.

An exemplary method of treating acne is disclosed herein. Exemplary methods include treating the at least one site of acne on the skin with a plasma and then applying the antimicrobial agent to one or more sites of the acne.

Exemplary methods of applying moisturizing agents to the skin are disclosed herein. Exemplary methods include applying a plasma to the skin surface to open the pores of the skin; Then applying a moisturizing agent to the surface of the skin; And transporting the moisturizing agent to the desired depth through the pores.

An exemplary method of applying a cosmetic to the skin is disclosed herein. Exemplary methods include applying a plasma to the skin surface to open the pores of the skin; Then apply cosmetics to the skin surface; And transporting the moisturizing agent to the desired depth through the pores.

These and other features and advantages of the present invention will be better understood in the light of the following description and the accompanying drawings.
Figure 1 is an exemplary illustration of a skin layer;
Figure 2 shows an exemplary transdermal delivery system for opening pores in the skin and delivering or transferring molecules through the skin;
Figure 3 shows another exemplary transdermal delivery system for opening pores in the skin and transferring or transferring molecules across the skin
Figure 4 illustrates a third exemplary transdermal delivery system for opening pores in the skin and delivering or transferring molecules across the skin;
Figure 5 illustrates a subsequent exemplary transdermal delivery system for opening pores in the skin and transferring or transferring molecules across the skin; And
6 is a plan view of the electrode of Fig.

FIG. 2 illustrates an exemplary embodiment of a percutaneous delivery system 200 for delivering or transferring molecules through pores open to the skin 220 and openings to the skin 220. The exemplary percutaneous delivery system 200 includes a non-thermal plasma generator 201 that includes a high-voltage tubular electrode 202 and a borosilicate glass tube 204. Plasma generator 201 is a floating-electrode dielectric barrier discharge (DBD) plasma generator that generates a plasma "jet"

The plasma generator 201 includes a gas supply 215. Exemplary gases that can be used in the plasma jet supply comprises a _{He, He + O 2, N} 2, He + N 2, Ar, Ar + O 2, Ar + N 2, and the like. A gas obtained from the evaporation of the liquid solution may also be used. Examples of vaporized liquids may include water, ethanol, organic solvents, and the like. These vaporized liquids may be mixed with the additive compound. The evaporated liquid and additives may be used together with or without gas at various concentrations as identified above. The plasma generator 201 includes a power supply not shown. The power supply is a high voltage supply and can be configured in a variety of different wave forms such as, for example, constant, ramp-up, ramp-down, pulse, nanosecond pulse, microsecond pulse, square, sinusoidal, random, in-phase, out-of-phase, and so on. In some exemplary embodiments, the power supply was a microsecond pulse power supply. The plasma 206 is generated by applying an AC polarity pulse voltage. The voltage is about 20 kV (peak-to-peak) at a pulse width of about 1 - 10 μs and a power density of 0.1 - 10 W / cm ² with a rise time of 5 V / ns at an operating frequency of 50 Hz to 3.5 kHz. . During operation, the plasma jet 206 is in direct contact with the skin 220.

Plasma develops across the skin a voltage potential that leads to intracellular and intercellular perforation, allowing the electric field to reach the skin and deposit electrical charges. In the exemplary system described herein, the working gas of the plasma jet 206 was a helium at a flow rate of 3 slm (liters per standard minute); The operating frequency was 3500 Hz with a pulse width of 1 μs and an operating period of 100%. The space between the jet nozzle and the skin to be treated was kept at 5 mm. The use of helium gas reduced the plasma temperature and increased the working distance to the skin 220 compared to air. As described above, the plasma piercing is non-invasive since the plasma electrode is not in contact with the tissue or substrate to be treated.

In terms of intracellular perforation, the membrane penetration voltage of the fluid lipid bilayer membrane reaches about 0.2 V or higher. The membrane through voltage charges the lipid bilayer membrane and causes a rapid, local structural rearrangement in the membrane and a transition to a membrane structure filled with water that punches through the membrane to form an "aqueous path" or "pore ". An aqueous pathway or pore allows an overall increase in ion and molecular transport. The membrane penetration voltage is believed to produce a primary membrane "pore" of a minimum radius of about 1 nm. In addition, the applied electric field causes rapid polarization changes that modify the mechanically unrestrained cell membranes (e. G., Suspended vesicles and cells) and cause ionic charge redistribution, which is governed by electrolyte conductance.

Electrical pulses used to generate the plasma jet 206 also cause intercellular perforation. SCs of about 15 - 25 μm thickness are the most electrically resilient parts of the skin. Application of electrical pulses used to generate the plasma jet 206 results in transcutaneous voltage over about 50 V to about 100 V, which causes perforation of the multilamellar bilayer in the SC. At these applied levels of transdermal voltage, perforation of the cell lining of the sweat glands and hair follicles also occurs.

When removing the plasma source from the treated area, the pores tend to close again and thus the process is reversible. Some of the pores remain open for extended periods of time, during which molecules can continue to cross cell membranes through diffusion. In some embodiments, it has been found that the pores remain open for less than about 5 minutes. The experimental results demonstrate that 10 kDa dextran molecules applied to the plasma treated region when transported within 0 to about 5 minutes are transported through the open pores in the SC. After 5 minutes, the 10 kDa dextran molecule no longer passes through the SC.

When an electric pulse is applied to the skin, the absorbed energy may cause local heating and damage to the skin. Energy above 50 J / cm ² deposited on intact skin causes secondary burns and thermal damage to underlying intact skin. One way to overcome this problem is to repeatedly apply short duration pulses that allow the same amount of energy to cause damage to be delivered without causing local heating and skin damage. In some embodiments, the energy deposited on intact skin is about 25 J / cm &lt; ² &gt; And in some embodiments, the energy deposited on the intact skin is less than about 10 J / cm &lt; ² &gt; And in some embodiments, the energy deposited on the intact skin is less than about 5 J / cm &lt; ² &gt; And in some embodiments, the energy deposited on the intact skin is less than about 3 J / cm &lt; ² &gt; . However, when treating a wound, the energy can be increased to 500 J / cm ² without causing burns, for example. In some embodiments, an energy in the range of 500 J / cm &lt; ² &gt; can be used to coagulate blood.

In addition, damage to the skin can result from localized plasma microdischarge, also known as "streamer ", which occurs with non-uniform electric fields. This problem can be overcome by creating a uniform electric field. In some embodiments, helium gas may be used as the gas to be supplied to the plasma generator 201. It has been found that the use of helium provides a uniform plasma field and minimizes streamers. In addition, nanosecond pulsed power supplies provide a more uniform plasma field and thus provide less pain and / or damage to the skin. In addition, skin damage can be avoided by reducing the power level, frequency, duty cycle and pulse duration of the power supply and by increasing the space between the plasma electrode and the skin to be treated.

Once the plasma is applied to cause plasma perforation and when the plasma generating mechanism 206 is turned off, the aqueous path multi-lamella system remains open for a period of up to about several minutes to several hours.

Other types of plasma generators may be used for transdermal delivery systems, such as nanosecond pulsed DBD plasma, microsecond pulsed DBD plasma, sinusoidal DBD plasma, resistive barrier discharge plasma, surface DBD plasma, Or a 2-D or 3-D array of DBD plasma jets operating under controlled duty cycles spanning 1-100%. It is important to note that not all plasma generators can be used to successfully cause perforation. Examples of plasma generators are thermal plasmas, gliding arc discharges, DC hollow cathode discharges, positive or negative corona generators, and plasma tron generators that are not suitable for plasma drilling. These plasma generators do not carry conduction currents that cause thermal damage, muscle contraction and pain, or they do not transfer sufficient charge to the substrate being treated, there is no or very weak applied electric field, and therefore there is no induced perforation.

A suitable plasma generator has a dominant current that is a displacement current at low power and / or high frequency. Displacement currents have units of current density and have associated magnetic fields, as does conduction currents, but this is not a current in the moving charge, but a time-varying electric field. The electric field is applied to the skin by an insulated electrode that is not in contact with the skin. Because the electrodes are insulated and are not in contact with the skin, there is no conduction current flow into the skin that can cause thermal damage, muscle contraction, and pain associated with electroporation.

In the larger treatment area, electrode configurations composed of multiple plasma jets or larger area flat electrodes (not shown) may be used. In the case of a more complex 3D surface, a controlled plasma module (not shown) can move around the surface to be exposed to the plasma, which can be placed on a stationary target or movable stage. In some embodiments, one or more plasma jets may be attached to a robotic arm programmed to move in a manner that exposes one or more target regions to a plasma plume or jet.

In addition, in some embodiments, the plasma generator 201 may be combined with a biomolecule / drug delivery system, wherein the molecules are treated by needle-free injection, evaporation, spraying, or misting, Lt; / RTI &gt; area. In some embodiments, this may aid pretreatment of the surface.

And reducing the plasma temperature, and in some embodiments it is essential to promote skin penetration, leading to a plasma perforation, He, Ar, Ne, Xe or the like, air, or other gases, a small percentage (0.5% -20%), such as O ₂ And a mixture of inert gases having N ₂ and a mixture of inert gas having a vaporized liquid comprising water, DMSO, ethanol, isopropyl alcohol, n-butanol, with or without additives, etc., is advantageous to generate a non-thermal plasma .

FIG. 3 illustrates another exemplary percutaneous delivery system 300. The transdermal delivery system 300 includes a plasma generator 301. The plasma generator 301 includes an electrode 302 at a first end and a high voltage wire 303 connected to a high voltage power supply (not shown) at a second end. A suitable high voltage supply has been described above. In some exemplary embodiments, the power supply was a nanosecond pulse power supply. Plasma 306 was generated by applying an alternating polarity pulse voltage with a nanosecond duration pulse. The applied voltage is about 40 - 500 ns a pulse width of (a single pulse to the 20 kHz) 0.5 - 1 kV / in ns was held with a rise time of 0.01 - at a power density of 100 W / cm ² about ~ 20 kV (peak -To-peak). &Lt; / RTI &gt; The dielectric barrier 304 is located under the high voltage electrode 302. In addition, the high voltage electrode 302 is located within the housing 305. The plasma generator 301 is a non-thermal dielectric barrier discharge (DBD) generator. The plasma 306 is generated by the plasma generator 301. Figure 3 also includes a skin 320. The skin or tissue acts as a second electrode, can be grounded, or it can be floating ground. The plasma 306 is in direct contact with the skin 320. In the exemplary experimental results disclosed herein, the skin 320 is pig skin.

Direct plasma 306 is generated by applying an alternating polarity pulse voltage to electrode 302. The applied voltage has a pulse width of about 1 - 10 μs (100 Hz to 30 kHz) with a magnitude of about 20 kV (peak-to-peak). The power supply (not shown) was a variable voltage and variable frequency power supply. A 1 mm thick transparent quartz slide was used as the dielectric dielectric barrier 304 and covers the electrode 302. The electrode 302 was a 2.54 cm diameter copper electrode. The discharge gap between the dielectric barrier 304 and the pig skin 320 is about 4 mm +/- 1 mm. In some experiments, the pulse waveform has an amplitude of about 22 kV (peak-to-peak), a duration of about 9 μs with a rise time of about 5 V / ns. The discharge power density was about 0.1 W / cm ² to 2.08 W / cm ² . J / cm ² The plasma treatment capacity was calculated by multiplying the plasma discharge power density by the plasma treatment duration.

In addition, the indirect plasma 406 is generated by the plasma generator 401. The plasma generator 401 is similar to the plasma generator 301 except that the plasma generator 401 includes a metal mesh 330 for filtering the plasma 406. Metal mesh 300 prevents the passage of charged ions and electrons, but neutral species allows passage and contact with the skin. A neutral species may be referred to as "afterglow ".

FIG. 5 is a schematic diagram of another exemplary embodiment of percutaneous delivery system 500. FIG. 6 is a top view of the electrodes of the transdermal delivery system 500. FIG. Transdermal delivery system 500 includes a plurality of DBD jets. The exemplary transdermal delivery system 500 has an array of honeycomb DBD jets; However, many different configurations may be used, for example, linear, triangular, square, pentagonal, hexagonal, octagonal, and the like.

The DBD jets have glass tubes 504A, 504B, 504C, 504D, 504E, 504F and 504G. The metal electrode 502 includes a plurality of cylindrical openings 502A, 502B, 502C, 502D, 502E, 502F, and 502G that receive corresponding respective glass tubes 504A, 504B, 504C, 504D, 504E, 504F, . Optionally, multiple metal electrodes may be used. The metal electrode 502 may have an insulating sheath (not shown) to prevent shock. The metal electrode 502 is connected to a high voltage supply as described above.

The DBD jets have gas flow inlets located at the first end and have plasma jets 516A, 516B, 516C, 516D, 516E, 516F and 516G on the other side. As described above, the gas may be, for example, He, Ar, Ne, Xe, air, He + air, Ar + air, Ne + air, Xe + air, In addition, each glass tube 504A, 504B, 504C, 504D, 504E, 504F and 504G has an inlet 508A, 508B, 508C, 508D, 508E, 508F and 508G located along the glass tube to receive the vaporized liquid additive . These inlets may be located above or below the electrode 502. An exemplary transdermal delivery system 500 utilizes skin as a ground electrode.

In the various experiments described herein, some embodiments of the transdermal delivery system employ the direct plasma generator 201 described in the aspect of Figure 2, some using the direct plasma generator 301 described in the aspect of Figure 3 And partly uses the indirect plasma generator described in the aspect of Fig.

In the exemplary embodiment of Figures 2 and 3, the skin 220 is directly exposed to a plasma 206 containing charged species, including energetic electrons, neutral and negative, and positive ions. Similarly, an electrical discharge with the direct plasma generator 301 takes place between the dielectric barrier 304 and the skin 320 and direct skin to the charged particles, including active electrons, neutral reactive species and negative and positive ions Exposed.

Indirect plasma generated by the plasma generator 401 utilizes a grounded copper mesh (16 x 16 mesh size and 0.011 "wire diameter and 0.052" aperture size) placed between the high voltage electrode and the skin, Prevent contact with the skin surface.

Experiment result

Experimental results and processes are based on plasma treatment of pig skin to carry dextran molecules tagged with fluorescent dyes, or proteins tagged with fluorescent dyes or nanoparticles tagged with fluorescent dyes through the skin layer, where several experiments have been performed. The transport of dextran molecules of various sizes has demonstrated the feasibility of using plasma for transdermal delivery of molecules of different size, polarity and physicochemical properties.

Porcine skin with intact stratum corneum (SC) from the ears and abdomen has been used and includes both full (not saliva) and partial (fat) skin. Skin was kept at -80 ° C until treatment. On the day of treatment, the skin was thawed to room temperature and stored for one hour in a humid box. Prior to the application of the plasma, the hair was removed with a hair clipper and the skin was shaved. The skin was washed with soap and dried with a paper towel. The skin from the ear was cut into 1 "x 1" pieces and the skin from the abdomen was cut into 2 "x 2" pieces. The pieces of skin were kept on a wet paper towel in a wet box to maintain constant humidity.

Lysine fixability fluorescently tagged dextran molecules with molecular weights of 3, 10, 40 and 70 kDa were also used. Dextran does not diffuse freely through the skin by itself and has been used as a probe to determine the method and apparatus for the plasma-induced perforation ("plasma perforation ") process claimed and described herein. In each experiment, dextran molecules were reconstituted to a concentration of 5 mg / ml in deionized water.

In some experiments, pig skin was treated with a specific heat DBD plasma for a maximum period of about 3 minutes and the following plasma power source parameters were changed: the frequency (Hz) was varied from about 100 to about 3500 Hz, the pulse duration was about 1 to about 10 μs; The duty cycle was varied from about 1 to about 100%, and the treatment time was from about 0.5 to about 3 minutes.

In some experiments, 40 μL of dextran suspension was applied to the skin immediately after plasma treatment. In some experiments, the skin was plasma treated for about 1 minute, followed by a dextran solution, and then the target area was treated with plasma for about another minute. In some experiments, dextran solution was applied to the skin and treated with plasma for about 1 minute. In various experiments, the treated skin is allowed to interact with the dextran solution for 15, 30, 45 or 60 minutes in the dark.

After treatment, 5 - 10 mm punch biopsy samples were obtained from the control and plasma treated samples. Biopsy samples were immediately immersed in 10% neutral buffered formalin and stored at 4 ° C. Biopsy specimens for histological analysis were prepared using paraffin embedding followed by hematoxylin and eosin (H & E) staining or freeze cutting. A 10 μm slice was obtained perpendicular to the skin surface and placed on a glass microscope slide. Morphological and penetration depth analyzes were performed on EVOS reverse phase fluorescence enabled microscope (AMG Microscope).

Experimental results demonstrate that the nonthermal plasma induces perforation in intact pig skin without visible thermal damage to the underlying skin. In addition, there is evidence that a 3 kDa dextran molecule (1 nm hydrodynamic radius) has penetrated the skin to an average depth of 500 μm. A dextran molecule (2 nm hydrodynamic radius) of 10 kDa migrated to an average depth of 200 μm. These and more detailed results are provided in Table I below.

The second column of Table I indicates the type of plasma source used. The third column indicates the molecular weight of the dextran molecule. The fourth through sixth columns confirm the power supply settings for the plasma generator for a particular experiment. The seventh column indicates the time (if any) that the treated area was exposed to the plasma prior to applying the solution of dextran molecule to the treated area. Similarly, the eighth column indicates the time (if any) that the treated area was exposed to the plasma after the solution of the dextran molecule was applied to the treated area. The ninth column indicates the amount of time left in the treated area of the solution after plasma treatment and the tenth column indicates the average penetration depth of dextran molecules into the skin.

An exemplary DBD jet plasma generator shown in Figure 2 using helium gas as input is identified as "He DBD jet ". The DBD plasma generator shown in Fig. 3 is identified as "air DBD ".

<Table I>

The depth of penetration of fluorescently tagged dextran molecules for different plasma configurations and treatment modalities

The average depth of SC is about 10 [mu] m to 20 [mu] m. Thus, all of the above experimental results demonstrate that plasma perforation is successful in delivering molecules through SC, which is a major barrier to transdermal delivery.

In addition, reducing the pulse duration of a He DBD jet causes an increase in penetration depth in the skin. Increasing the plasma frequency also increased penetration depth into the skin. Increasing the working cycle also increased penetration depth into the skin. It has also been found that a short plasma treatment time (about 1 minute) results in a larger penetration depth into the skin. In addition, an increased penetration depth for high molecular weight dextran was observed at longer pulse durations.

Further, the use of a DBD jet plasma generator for plasma treatment results in an average higher penetration depth than a plasma generator generating a plasma using a conventional DBD. In addition, penetration depth is limited to the epidermis when using plasmas generated by plasma generators with normal DBD electrodes at longer pulse durations and shorter operating cycles. Thus, the results can be used to control the penetration depth of the target molecule, for example, by varying the plasma treatment parameters including, for example, the type, frequency, duty cycle, pulse duration, plasma treatment time and application time to the skin of the plasma generator used .

Application of the subject molecules before or after plasma treatment yields a similar penetration depth after a similar plasma exposure time. Thus, application of a plasma prior to the application of a sensitive molecule, drug or vaccine may be accomplished by contacting the susceptible molecule, drug or vaccine with a susceptible molecule, drug or vaccine through a newly created pore without loss of activity or degradation of the susceptible molecule, drug or vaccine due to interaction with the plasma or the associated electric field Allows penetration of the vaccine.

Table II below identifies a list of exemplary compounds of molecular weight that can be delivered through the skin using plasma perforation. The charge of the compound is also included in the chart.

<Table II>

Molecules suitable for transdermal drug delivery

Although some of the molecules listed in the above table have a MW of less than 500 daltons and may be suitable for transdermal delivery without plasma perforation, plasma perforation may increase the delivery rate and efficiency of therapeutic doses of the molecule, The need can be reduced.

In addition, plasma perforation can be used to deliver albumin into the epidermis via SC. Experimental results have demonstrated that albumin (66 kDa) tagged with green fluorescence (fluorescence tag enables detection of the target molecule via standard fluorescent capable imaging technology) can be delivered percutaneously by treating the skin with plasma. In some experiments, the power supply was set to 200 ns and 20 kV and used with various pulses. Three pulses, five pulses, and ten pulses were applied to the skin, all causing penetration of SC, and 10 pulses delivered deeper than albumin over 3 pulses. Another set of experiments treated the skin with different voltages at a power setting of 200 ns and 5 pulses. 10 kV, 15 kV and 20 kV were used. Albumin penetrated through the skin at all voltages and caused the deepest penetration at 20 kV. In addition, treatments using a power source set to 100 ns and 30 seconds at various different frequency settings cause different penetration depths. 500 Hz, 1000 Hz, and 5000 Hz both cause penetration into the epidermis, resulting in the deepest penetration at higher frequencies. Experimental results indicate that albumin is localized to the epidermis prominently at a depth of up to about 75 to 100 μm.

In addition, plasma perforation can be used to deliver fluorescently tagged IgG (human immunoglobulin G) into the epidermis and dermis via SC. Experimental results demonstrate that IgG (115 kDa) can be delivered percutaneously by treating the skin with a microsecond pulse plasma. In some experiments, a power source set to 200 ns and 5 μs was used to treat the skin at various frequencies for 30 seconds and IgG was applied to the skin for a 60 minute hold time. 500 Hz, 1500 Hz, and 3500 Hz all cause penetration into the epidermis, resulting in the deepest penetration at higher frequencies. In some experiments, a power source set at 200 ns and 3500 Hz was used to treat the skin with various pulse durations for 30 seconds and IgG was applied to the skin for a 60 minute hold time. 1 μs, 3 μs, and 5 μs pulses were used, all causing penetration into the epidermis and causing the deepest penetration at high frequencies. Thus, the transfer depth of IgG through the microsecond pulsed plasma-induced portion is dependent on the PDP (application of the plasma to the skin followed by application of the molecule and subsequent application of the plasma to the skin) In contrast, PD (proportional to the plasma frequency in the application of the plasma to the skin followed by the molecule) mode. The PDP mode enhances the penetration of IgG more deeply into the skin than the PD mode. Experimental results showed that IgG was localized predominantly in the epidermis, but strong signals were determined in the dermis at about 400 to 600 μm.

Table III identifies a list of topical drugs currently applied to the skin. These topical drugs may be applied as gels or creams. In some exemplary methodologies, these drugs can be applied after plasma drilling without the need for a dirty gel or cream. In addition, plasma perforation can reduce the amount of time required to deliver a therapeutic dose of the drug. Moreover, application of the compound after the plasma treatment allows the topical drug to rapidly penetrate the SC without altering the composition. Since the methods disclosed herein do not alter the chemical composition, it may not be necessary to obtain FDA approval for a new drug or composition, or the rate of approval may be increased. In addition, the topical drug may be applied without the gel or cream (or as a small gel or cream). In addition, since the absorption rate increases, a small amount of drug may be required.

<Table III>

Common topical drugs

Table IV identifies a list of drugs currently used in transdermal drug delivery systems that can be administered with less time, or using plasma perforation without the need for dirty cream and gel. The advantages described above in terms of Table III also apply to the list in Table IV.

<Table IV>

Transdermal drug

Plasma drilling has many other practical applications. In some embodiments, plasma perforation can be used to increase penetration of bactericides, antimicrobial agents, surgical scrubs, and the like. Exemplary disinfectants, antimicrobials, and surgical scrubs are identified in Table V below.

<Table V>

Fungicides and Antimicrobial Agents

Increasing the penetration of antimicrobial agents, for example, increases the rate and efficacy of killing undesirable microorganisms in the skin deep layer. In addition, certain antimicrobial agents take a long time to penetrate cell walls, but plasma perforation increases permeability and shortens the killing time.

Plasma perforation can also be used to treat acne. Plasma perforations can open conventional perforations as well as surrounding pores and sterilize infected areas. Second, plasma perforation allows antimicrobial agents and other acne medicines to enter the pores. Thus, rather than taking drugs with serious side effects, plasma perforation can be used without side effects. In addition, plasma treatment may not need to be used daily and may be used to treat acne at predetermined intervals, such as once per week, several times per week, and the like. In some embodiments, the plasma treatment is only needed periodically.

Plasma punching can also be used to open the pores to reduce wrinkles and to transfer cosmetic-related materials such as collagen, BOTOX or other fillers into the skin. Table VI identifies exemplary cosmetics suitable for use with plasma perforations.

<Table 6>

Skin filler

Plasma perforation can be used to increase the uptake rate of the moisturizing agent thereby minimizing "sticking" associated with the non-fully absorbed moisturizing agent. Heavy moisturizers, which are generally not permeable to the skin, do so after plasma drilling. Heavy moisturizers suitable for use with the exemplary plasma perforations are identified in Table VII below.

<Table VII>

Heavy moisturizer

In some embodiments, the skin may be pre-conditioned to temporarily alter skin pH, moisturizing, temperature, electrolyte concentration, and the like. Pre-conditioning helps to maximize the rate and depth of penetration of the active ingredient through pore formation without compromising the skin.

In some embodiments, plasma perforation is used to achieve penetration of the surfactant to the epidermal depth of the skin, allowing for more effective release and removal of buried contaminants adhering to the skin, either alone or in combination with a hand- Can be used. Exemplary surfactants are provided in Table VIII below.

<Table VIII>

Powerful surfactant

In addition, plasma perforations can enable the use of chemically less aggressive surfactants and / or less concentration and volume. Exemplary less potent surfactant compounds are shown in Table IX below.

<Table IX>

Less powerful surfactant compounds

In some embodiments, plasma perforation can be used in combination with low levels of non-irritating chemical skin penetration enhancers to achieve synergistic penetration of the active agents, including antimicrobial agents, cosmetic ingredients, vaccines, or drugs. Examples of chemical enhancers include dimethyl sulfoxide, azone, pyrrolidone, oxazolidinone, urea, oleic acid, ethanol, liposomes. The molecular weight of the exemplary chemical penetration enhancer is shown in Table X below.

<Table X>

Skin penetration enhancer

As described above, plasma perforation can be used to move common topical drugs into the skin faster. The advantages of delivering conventional topical medicaments faster into the skin include maintaining a more stringent therapeutic concentration, eliminating the need for mixing topical drugs with other compounds such as dirty gels for proper absorption. The methodology eliminates the need for additional FDA approval or results in an increased approval rate.

In addition, in some embodiments, antioxidants designed to achieve an optimal balance of skin penetration performance and skin safety may be used to avoid overdose that causes mutagenic damage to the DNA of cells in an immune response or viable epidermis that is unfavorable And is delivered during plasma drilling to neutralize the oxidant contained in the plasma.

The combination of a non-thermal plasma gas, an electric field and a chemical oxidant with controlled programming over time of the adjustable parameter can achieve optimal results for the treatment of skin or biofilm.

Although many of the above exemplary methods relate to molecules, particles having similar molecular weights or equivalent diameters may also be transported across the skin layer. In some embodiments, nanoparticles, such as, for example, silver nanoparticles, silver ions, and other metals or polymeric nanoparticles are transferred into the pores in the skin that they are allowed to react to. Silver, copper and other metals are known to induce cell lysis and inhibit cell transduction. The introduction of silver and other metals in the form of nanoparticles increases the surface area capable of reacting with microorganisms and enhances the antimicrobial activity. Additionally, the introduction of nanoparticles that encapsulate molecules, vaccines, or drug targets after plasma perforation allows penetration of such molecules to a controlled depth and leads to controlled long-term release of the active agent within a particular area of the skin. Nanoparticles having a diameter of about 2 to about 400 nanometers, including quantum dots, nanotubes, and the like, can be moved across the skin using plasma perforation.

In a further experiment it was found that the penetration depth of the 3 kDa dextran molecule was directly proportional to the operating period of the applied voltage.

Experiments were performed using a helium DBD plasma jet with a 5 mm gap between the plasma jet and the skin surface. The skin was treated with plasma at 3500 Hz for 30 seconds with a microsecond pulsed power supply. As can be seen in Chart I, increasing the pulse duration of the applied voltage causes an increase in the penetration depth of the dextran molecule.

Another experiment demonstrated the penetration of nanoparticles after plasma treatment. Ex vivo pig-skin was treated with a plasma using a nanosecond pulsed power supply. 100 [mu] l of 50 nanometer fluorescent tagged silica nanoparticles in aqueous solution were applied to the treated area for 15 minutes to 1 hour. A biopsy specimen was obtained to obtain the slides treated with the cryostat to be photographed. At fixed pulse durations and fixed treatment times, it has been found that the depth of penetration increases as the frequency of the plasma increases. At a fixed frequency and fixed treatment time, the depth of penetration increased as the pulse duration of the plasma increased. At the fixed frequency and pulse duration of the fixed plasma, penetration depth increased with increasing treatment time. It was also found that a plasma treatment of 15 seconds would move the nanoparticles to a depth of about 175 μm and a 30 second treatment at 1 kHz could move the nanoparticles to a depth of about 222 μm. These results are shown below.

As can be seen in Chart II, an increase in the number of applied pulses leads to an increase in penetration depth. An increase in the volume of applied nanoparticles initially causes an increase in penetration depth and then saturates. Similarly, by increasing the concentration of applied nanoparticles, an increase in penetration depth is observed up to a certain depth and then saturated. The penetration depth is directly dependent on the applied pulse duration and the frequency of the pulse application. In addition, it has been found that the application of discontinuous nanosecond duration pulses achieves a similar or better result than successive applications. It has also been found that decreasing the pulse duration, increasing the frequency, or increasing the operating cycle increases the penetration depth into the skin. Longer pulse durations result in shallower penetration depths. Surprisingly, shorter treatment times result in larger penetration depths into the skin.

As can be seen in Chart III, similar to Chart II, an increase in the number of applied pulses leads to an increase in penetration depth of the applied molecule. An increase in the volume of applied molecules initially leads to an increase in penetration depth and then to saturation. Similarly, by increasing the concentration of applied molecules, an increase in penetration depth is observed up to a certain depth and then saturated. The penetration depth is directly dependent on the applied pulse duration and the frequency of the pulse application. In addition, it has been found that the application of discontinuous nanosecond duration pulses achieves a similar or better result than successive applications. It has also been found that decreasing the pulse duration or increasing the frequency and / or increasing the cycle duration increases the penetration depth into the skin. Longer pulse durations result in shallower penetration depths. Surprisingly, shorter treatment times result in larger penetration depths into the skin.

While an exemplary embodiment is depicted using the skin, any of the embodiments described are equally well suited to any tissue including epithelial tissue, mucosal epithelial tissue, muscle tissue, connective tissue, internal and external lining of organs, .

While the invention herein has been illustrated by the description of the embodiments and the embodiments have been described in considerable detail, it is not the intention of the applicant to limit such details or limit the scope of the appended claims in any way. Additional advantages and modifications will readily appear to those skilled in the art. Accordingly, the invention in its broader aspects is not limited to the specific details, representative apparatus, and illustrative embodiments shown and described. Thus, developments may be made from such details within the spirit and scope of the applicant's general concept.

Claims (38)

Opening one or more pores in the skin by applying a plasma field to the skin;
Applying to the skin surface a carrier having at least one molecule selected from one of cream, patch, gel, ointment, aerosol, or liquid and having a molecular weight greater than 500 Da;
Allowing one or more molecules to pass through the pores to a desired depth; And
Closing one or more pores
Wherein the molecule is delivered or transferred into the skin. The method of claim 1, wherein the carrier comprises molecules dissolved in solution. The method of claim 1, wherein the carrier comprises molecules in suspension. The method of claim 1, wherein the carrier comprises charged molecules. The method of claim 1, wherein the at least one molecule has a molecular weight of greater than 1000 Da. The method of claim 1, wherein the at least one molecule has a molecular weight of greater than 3000 Da. The method of claim 1, wherein the at least one molecule has a molecular weight of greater than 10,000 Da. The method of claim 1, wherein the at least one molecule comprises a moisturizing molecule. The method of claim 1, wherein the at least one molecule comprises a cosmetic molecule. 10. The method of claim 9, wherein the cosmetic molecule comprises a filler. 11. The method of claim 10, wherein the filler comprises collagen. 2. The method of claim 1, wherein the at least one molecule comprises a bactericidal molecule. The method of claim 1, wherein a plasma is applied to the skin surface, a carrier comprising the one or more molecules is applied to the skin surface for a predetermined amount of time, and after one or more molecules have passed through the pores produced by the first plasma application Wherein the plasma is applied to the skin surface. 14. The method of claim 13, wherein the time between application of the carrier containing one or more molecules and the second plasma treatment is from about 1 second to about 120 seconds. The method of claim 1, wherein the plasma field is generated by contact with the skin for about 1 second to about 120 seconds by a plasma generator that generates a continuous plasma. The method of claim 1, wherein the plasma field is generated by a plasma generator set at a pulse repetition frequency of about 2 to 20,000 Hz. The method of claim 1, further comprising setting the pulse duration of the plasma generator for plasma generation to between about 1 μs and about 10 μs. 2. The method of claim 1, further comprising setting the pulse duration of the plasma generator for plasma generation to from about 0.1 nanoseconds to about 500 nanoseconds. The method of claim 1, further comprising setting an operating period of the plasma generator for plasma generation to about 10 to about 100%. The method of claim 1, wherein the plasma is generated by a plasma generator and the plasma generator is a dielectric barrier discharge jet plasma generator. The method of claim 1 wherein the plasma is generated by a plasma generator and the plasma generator is a dielectric barrier discharge jet plasma generator and has a helium gas supply. The method of claim 1, wherein the plasma is generated by a plasma generator and the plasma generator is a dielectric barrier discharge plasma generator that uses ambient air to produce a plasma. The method of claim 1 wherein the plasma is generated by a plasma generator and the plasma generator has a microsecond pulsed power supply. The method of claim 1, wherein the plasma is generated by a plasma generator and the plasma generator has a nanosecond pulsed power supply. 25. The method of claim 24, wherein the nanosecond pulsed power supply applies plasma to the skin in multiple discrete pulses. 25. The method of claim 24, wherein the number of discontinuous pulses can range from about 1 to about 100 discrete pulses with a pulse duration of about 1 ns to about 500 ns. The method of claim 1, wherein the one or more molecules are moved to an average depth of about 30 to 600 μm. 3. The method of claim 1, wherein at least one molecule in the molecule is moved to an average depth of about 125 to 500 [mu] m. 3. The method of claim 1 wherein one or more molecules in the molecule are moved to an average depth of about 200 to 400 [mu] m. 2. The method of claim 1, wherein the at least one molecule is moved to an average depth of about 200 to 1000 [mu] m. The method of claim 1, further comprising preconditioning the skin prior to applying the plasma to the skin. 32. The method of claim 31, wherein the pre-conditioning comprises altering one or more of a skin pH, a moisturizing degree, a temperature or an electrolyte concentration. 32. The method of claim 31, further comprising applying a chemical skin penetration enhancer to the skin. 34. The method of claim 33, wherein the chemical skin penetration enhancer is one of dimethylsulfoxide, oleic acid, or ethanol. Opening one or more pores in the skin by applying a plasma field to the skin;
Applying at least one nanoparticle having a size of less than 600 nm to the skin surface;
Allowing at least one pore to transport the at least one nanoparticle through the at least one pore; And
Allowing the at least one nanoparticle to pass through at least one pore to a desired depth; And
Closing one or more pores
Wherein the nanoparticles are delivered or transferred into the skin. 36. The method of claim 35, wherein the at least one nanoparticle comprises silver, zinc oxide, titanium dioxide, silica, chitosan or a quantum dot. 37. The method of claim 35, wherein the at least one nanoparticle encapsulates at least one molecule, vaccine, or drug. Opening one or more pores in the skin by applying a plasma field to the skin;
Applying a topical or transdermal drug to the skin;
Allowing the percutaneous drug or topical drug to pass through at least one pore to a desired depth;
And closing at least one of the pores
Wherein the rate of penetration of the transdermal drug or topical drug is increased.

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